A novel role for myeloid endothelin-B receptors in hypertension

(1)

HAL Id: inserm-02439065

https://www.hal.inserm.fr/inserm-02439065

Submitted on 14 Jan 2020

HAL is a multi-disciplinary open access

archive for the deposit and dissemination of

sci-entific research documents, whether they are

pub-lished or not. The documents may come from

teaching and research institutions in France or

abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est

destinée au dépôt et à la diffusion de documents

scientifiques de niveau recherche, publiés ou non,

émanant des établissements d’enseignement et de

recherche français ou étrangers, des laboratoires

publics ou privés.

A novel role for myeloid endothelin-B receptors in

hypertension

Alicja Czopek, Rebecca Moorhouse, Léa Guyonnet, Tariq Farrah, Olivia

Lenoir, Elizabeth Owen, Job van Bragt, Hannah Costello, Filippo

Menolascina, Véronique Baudrie, et al.

To cite this version:

Alicja Czopek, Rebecca Moorhouse, Léa Guyonnet, Tariq Farrah, Olivia Lenoir, et al.. A novel role

for myeloid endothelin-B receptors in hypertension. European Heart Journal, Oxford University Press

(OUP): Policy B, 2019, 40 (9), pp.768-784. �10.1093/eurheartj/ehy881�. �inserm-02439065�

(2)

..

A novel role for myeloid endothelin-B

receptors in hypertension

Alicja Czopek

1†

, Rebecca Moorhouse

1†

, Le´a Guyonnet

2

, Tariq Farrah

1

,

Olivia Lenoir

2

, Elizabeth Owen

1

, Job van Bragt

1

, Hannah M. Costello

1

,

Filippo Menolascina

3,4

, Ve´ronique Baudrie

2

, David J. Webb

1

, David C. Kluth

4

,

Matthew A. Bailey

1‡

, Pierre-Louis Tharaux

2,5‡

, and Neeraj Dhaun

1,2

*

‡

1

BHF Centre for Cardiovascular Science, The Queen’s Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UK;2

Paris

Cardiovascular Research Centre - PARCC, Institut National de la Sante´ et de la Recherche Me´dicale (INSERM), Paris 75015, France;3School of Engineering & SynthSys, Institute

for Bioengineering, Centre for Synthetic and Systems Biology, University of Edinburgh, Edinburgh EH9 3BD, UK;4

MRC Centre for Inflammation Research, The Queen’s Medical

Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UK; and5Paris Descartes University, Sorbonne Paris Cite´, Paris 75006, France

Received 30 March 2018; revised 4 September 2018; editorial decision 10 December 2018; accepted 10 December 2018; online publish-ahead-of-print 17 January 2019

Aims Hypertension is common. Recent data suggest that macrophages (M/) contribute to, and protect from, hyperten-sion. Endothelin-1 (ET-1) is the most potent endogenous vasoconstrictor with additional pro-inflammatory proper-ties. We investigated the role of the ET system in experimental and clinical hypertension by modifying M/ number and phenotype.

... Methods

and results

In vitro, M/ ET receptor function was explored using pharmacological, gene silencing, and knockout approaches. Using the CD11b-DTR mouse and novel mice with myeloid cell-specific endothelin-B (ETB) receptor deficiency

(LysMETB-/-), we explored the effects of modifying M/ number and phenotype on the hypertensive effects of ET-1,

angiotensin II (ANG II), a model that is ET-1 dependent, and salt. In patients with small vessel vasculitis, the impacts of M/ depleting and non-depleting therapies on blood pressure (BP) and endothelial function were examined. Mouse and human M/ expressed both endothelin-A and ETB receptors and displayed chemokinesis to ET-1.

However, stimulation of M/ with exogenous ET-1 did not polarize M/ phenotype. Interestingly, both mouse and human M/ cleared ET-1 through ETB receptor mediated, and dynamin-dependent, endocytosis. M/ depletion

resulted in an augmented chronic hypertensive response to both ET-1 and salt. LysMETB-/-mice displayed an

exag-gerated hypertensive response to both ET-1 and ANG II. Finally, in patients who received M/ depleting immuno-therapy BP was higher and endothelial function worse than in those receiving non-depleting therapies.

... Conclusion M/ and ET-1 may play an important role in BP control and potentially have a critical role as a therapeutic target in

hypertension.

䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏

Keywords Myeloid cell

_•

Endothelin

_•

Hypertension

Introduction

Arterial hypertension is a major risk factor for atherosclerosis, coron-ary artery disease, stroke, and chronic kidney disease, and is a

prominent contributor to death worldwide.1It is estimated that a quarter of the world’s adult population is hypertensive and this num-ber is projected to rise to nearly 30% by 2025.2By age 70 years, 70% of the US population have hypertension. However, despite the

* Corresponding author. Tel:þ44 131 242 9215, Fax: +44 131 242 6779, Email:bean.dhaun@ed.ac.uk

†

These authors are joint first authors.

‡

These authors are joint last authors.

VC_{The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.}

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

European Heart Journal (2019) 40, 768–784

BASIC SCIENCE

doi:10.1093/eurheartj/ehy881

_Hypertension

(3)

..

frequency of hypertension, its cause in the majority of adults is unknown.

Hypertension is complex, with no single mechanism—sodium re-tention, renin release, and increased vascular tone—entirely explain-ing the blood pressure (BP) rise. The past 50 years have seen growexplain-ing evidence implicating the immune system.3Recent data suggest that macrophages (M/) contribute to, and protect from, hypertension. Early studies in the spontaneously hypertensive rat4found a correl-ation between the distribution of sub-endothelial M/ and endothelial function, and that treatment with an angiotensin converting enzyme inhibitor improved endothelial function and reduced the number of vascular M/. Furthermore, many models of hypertension—angioten-sin II (ANG II), high salt—are associated with renal accumulation of M/.5,6Despite these and many other observational studies, few have attempted to modify M/ phenotype/number to examine their role in hypertension.7–10

Endothelin-1 (ET-1) is the most potent endogenous vasocon-strictor.11 Its production is triggered by multiple stimuli includ-ing ANG II and pro-inflammatory cytokines.12,13 ET-1 acts by binding to two distinct receptors, the endothelin-A (ETA) and

the endothelin-B (ETB) receptors.14,15 ETA and ETB receptors

on vascular smooth muscle cell (VSMC) mediate the vasocon-strictor effects of ET-1.16 ETBreceptors are also found on

vas-cular endothelial cells (EC) where their activation results in dilation.17,18 Production of vascular ET-1 is increased in some but not all animal models of hypertension.19 Interestingly, ET antagonism can blunt BP elevation in ANG II infused rats sug-gesting that in this model, ET-1 largely mediates the hyperten-sive effects of ANG II.20,21 Importantly, a number of clinical studies have gone on to show the effectiveness of both select-ive ETA and mixed ETA/B antagonism in lowering BP in

hyper-tension.22–24

ET-1 is considered to be pro-inflammatory. ETAreceptor

activa-tion is crucial in mediating the ANG II-induced infiltraactiva-tion of renal cortical T cells.25In both models of aldosterone-induced hyperten-sion and diabetic nephropathy, selective ETAreceptor antagonism

reduced M/ infiltration,26,27and in the diabetic model also reduced fibrosis.27_{However, the effects of ET-1 on M/ biology are not well}

studied. In the current studies, we explored the role of M/ and monocytes in mediating the pro-hypertensive effects of ET-1 and ANG II.

Methods

SeeSupplementary material online.

Results

Macrophages possess endothelin

receptors but are not polarized by

endothelin-1

We first found that mouse bone marrow-derived macrophages (BMDM) possess both ETAand ETBreceptors (ETB> ETA) although

in relatively lower amounts than seen in VSMC and EC, respectively (seeSupplementary material online,Figure S1). To examine the role of the M/ ETBreceptor in greater detail, we generated novel mice

deficient in ETBon myeloid cells alone (LysMETB-/-). These mice

showed no differences in baseline vascular function or circulating im-mune cells compared with littermate controls (see Figure 1 and

Supplementary material online,Figures S2–S4).

We then explored the ability of ET-1 to polarize BMDM. Increasing concentrations of ET-1 (10–104pg/mL) were unable to polarize BMDM to a classical (M1) or alternative (M2) phenotype ei-ther alone or in combination (see Figure2andSupplementary mater-ial online,Figure S5). Furthermore, neither co-stimulation with LPS and ET-1 nor LPS stimulation following ET-1 priming, in the presence or absence of ET receptor blockade, augmented the BMDM re-sponse to LPS (seeSupplementary material online,Figure S6).

BMDM stimulation with LPS/INFc (but not IL-4/IL-13) increased the media concentration of ET-1 at 24 h (see

Supplementary material online, Figure S7), an effect that was blocked by phosphoramidon, an inhibitor of endothelin convert-ing enzyme (ECE) which catalyzes the conversion of big ET-1 to the mature peptide. Thus, this increase in ET-1 likely represents de novo production.

Macrophages demonstrate chemokinesis

to endothelin-1

Despite the lack of polarization by ET-1, BMDM demonstrated che-mokinesis to ET-1. This effect was more apparent at higher concen-trations of ET-1 and no different to MCP-1 at ET-1 103pg/mL and 104pg/mL. BMDM chemokinesis to ET-1 was blocked by both

Translational perspective

Hypertension is a costly global health problem, and an important risk factor for the development and progression of chronic

kidney disease. Its aetiology remains unclear in most adults. Here, the data provided suggest that the immune and endothelin

systems play important roles in blood pressure regulation and provide a rational basis for further investigation into the

modulation of these pathways. These studies may encourage industry to take a lead in this relatively orphan area, potentially

resulting in a more rational prescribing of endothelin receptor antagonists for hypertension that has developed as part of a

multi-system inflammatory disease, with these agents potentially affording broader cardiovascular protection. These studies

may also help inform the design of novel antihypertensive therapies.

Myeloid endothelin-B receptors in hypertension

769

(4)

..

.

selective ETA(BQ123) and selective ETB(BQ788) receptor

antagon-ism (Figure3A). In terms of chemokinetic ability, LysMETB-/-BMDM

had a blunted response to incremental concentrations of ET-1 com-pared with control BMDM, although the response to MCP-1 was maintained (Figure3B).

Macrophages demonstrate

endothelin-B-mediated endothelin-1 uptake

BMDM were then exposed to ET-1 10 pg/mL in their media. Serial assay of the media for ET-1 showed a gradual reduction over a 24 h period with the concentration at 24 h being approximately 60% that at baseline (Figure 4A). This reduction in media ET-1 was seen when BMDM were exposed to increasing concentrations of ET-1 (Figure4B) and was not explained by degradation of the peptide over the 24 h period (seeSupplementary material online,Figure S8). The fall in ET-1 was prevented by both selective antagonism of the ETBreceptor (but

not ETA) as well as inhibition of dynamin-dependent endocytosis (see

Figure 4C andSupplementary material online,Figure S9), supporting BMDM uptake of ET-1 through ETBreceptor-mediated endocytosis.

Neutral endopeptidase (NEP) inhibition had no effect. Additionally, mixed ETA/Breceptor antagonism was no different to selective ETB

blockade alone (seeSupplementary material online,Figure S10). Further data to support the role of the M/ ETBreceptor in

clear-ing ET-1 were provided usclear-ing an ETBgene silencing approach. M/

EDNRB knockdown again prevented ET-1 uptake by BMDM, an effect that was similar in magnitude to that seen with pharmacological ETB

receptor antagonism (Figure4D). As ETBreceptors are also present

on VSMC, we assessed their ability to remove ET-1 in vitro. VSMC did not remove ET-1 from their surrounding media (Figure4E). BMDM

from LysMETB-/-and controls were also exposed to ET-1 for 24 h. As

previously seen, control BMDM removed ET-1 from their medium, an effect that was blocked by BQ788. Medium from LysMETB

-/-BMDM showed no difference in ET-1 concentration at 24 h com-pared with baseline and there was no effect of pre-treatment with BQ788 (Figure4F). To further support our hypothesis of M/ ET-1 uptake, we exposed BMDM to fluorescent ET-1. Our results sug-gested a statistically significant (P < 0.01) increase in BMDM intra-cellular fluorescence following treatment with ET-1 (Figure4G). Uptake of fluorescence was blocked by selective blockade of the ETBreceptor and absent, as expected, in BMDM from LysMETB

-/-mice. Furthermore, this uptake process occurred rapidly (Figure4H) and reached a steady state within 60 min.

Macrophages modify vascular

contractility

The functional importance of M/ ETBreceptor-mediated ET-1

up-take is demonstrated in Figure5. Here, we infused M/ into the myog-raphy bath 15 min prior to infusing ET-1. Increasing number of M/ significantly attenuated the vasoconstrictor actions of ET-1 (P < 0.001 vs. control); this effect was lost when the M/ were pre-treated with selective ETBreceptor antagonism or when LysMETB-/-BMDM were

infused. There was no effect of pre-treating M/ with a selective ETA

receptor antagonist. M/ pre-treatment did not alter the contractile response to KCl. Again, these findings are in keeping with a rapid binding of ET-1 to M/ ETB that blunts its functional effects.

Additionally, and as expected, ET-1 increased oxidative stress in mes-enteric vessels; this effect was attenuated by M/ and attenuation was dependent on an unblocked ETBreceptor.

Figure 1Characterization of the endothelin system in LysMETB-/-. (A) qRT-PCR analysis of EDNRB (endothelin-B), EDNRA (endothelin-A), and

EDN1 (endothelin-1) expression in bone derived M/. (B) EDNRB (endothelin-B) expression in peritoneal M/ (left panel), bone marrow-derived M/, T and B cells (middle panel), and plasma endothelin-1 (right panel). Data are expressed as mean ± standard deviation and comparisons were made with unpaired t-tests and for (B) middle panel, Holm–Sidak was used to correct for multiple comparisons; adjusted P-values are shown.

770

A. Czopek et al.

(5)

..

Human and mouse macrophages show

similar responses to endothelin-1

As in mouse BMDM, human M/ showed expression of both the ETA and ETB receptor (see Supplementary material online,

Figure 11A). Similarly, human M/ were not polarized to a classical or alternative phenotype by ET-1 (see Supplementary material online, Table S1 and Figure S11B) but showed

evidence of ETB receptor-mediated ET-1 uptake and

Figure 2In vitro stimulation with endothelin-1 does not polarize M/ to a classical or alternative phenotype. (A) Bone marrow-derived M/ produc-tion of TNFa, IL-6, and IL-10 after 24 h stimulaproduc-tion with endothelin-1 (10–104pg/mL), LPS/INFc, IL-4/IL-13, and in untreated controls. Data (mean-± standard deviation; n = 4 per group) were compared by two-way analysis of variance, with main effects of treatment (P < 0.0001), production (P = 0.186), and the interaction (P = 0.0006). Multiple comparisons were made to compare the effect of each treatment against control (media alone), with a family P-value of 0.01; an adjusted P-value is shown. (B) Bone marrow-derived M/ mRNA production of a range of M1 (left) and M2 (right) markers following 24 h stimulation with endothelin-1 (10 pg/mL and 104pg/mL). Data (mean ± standard deviation; n = 4 per group) were compared by two-way analysis of variance (main effects of treatment, of gene product and the interaction were all P < 0.0001). Multiple comparisons were made to compare the effect of each treatment on mRNA production against control (media), with a family P-value of 0.01. Adjusted P-values are shown. (C) Western blot analysis demonstrating endothelin-1, either alone or in combination with LPS/INFc, did not stimulate production of caspase 1 in mouse M/. (D) Endothelin-1 alone (media) or in combination with LPS/INFc, did not stimulate production of IL-1b by mouse M/.

771

(6)

..

.

chemokinesis to ET-1 and (seeSupplementary material online,

Figure S11Cand D).

Macrophage depletion augments the

pressor response to endothelin-1 but

not angiotensin II

To understand the role of M/ in the pressor response to ET-1, we administered incremental doses of intravenous (i.v.) ET-1 to

CD11b-DTR mice given diphtheria toxin (DT) and to controls. M/ depletion per se was not associated with a difference in baseline mean arterial pressure (MAP) (Figure 6A) or a shift in vasoconstrictor-vasodilator capacity of conduit or resistance vessels (see

Supplementary material online,Figure S12). Administration of ET-1 following M/ depletion resulted in an exaggerated hypertensive re-sponse compared with controls (Figure6B) with a greater maximal change in mean arterial pressure (MAP) in M/-deficient mice (Figure6C). At a dose of ET-1 1 nmol/kg the maximal change in MAP Figure 3Macrophage demonstrate chemokinesis towards endothelin-1. (A) Bone marrow-derived M/ chemokinesis in response to increasing doses of endothelin-1 in the presence or absence of selective endothelin-A (BQ123) or endothelin-B (BQ788) antagonism; MCP-1 was used as a positive control. The number of M/ per high powered field (mean ± standard deviation; n = 6 mice per group) was compared by two-way analysis of variance, with main effects of endothelin-1 or MCP-1 treatment (P < 0.0001) and receptor antagonism (P < 0.0001) and the interaction (P < 0.0001). Effects within rows were compared, using one family per row and a family P-value of 0.01. Adjusted P-values are shown. (B) Chemokinesis of bone marrow-derived M/ in response to endothelin-1 or MCP-1. Bone marrow-derived M/ were isolated from LysMETB-/-(open circles; n = 6) and

con-trol (closed circles; n = 6) mice. Data were compared by two-way analysis of variance, with main effects of endothelin-1 or MCP-1 treatment (P < 0.0001) and genotype (P < 0.0001) and the interaction (P < 0.0001). Between genotype comparisons were made and adjusted P-values are shown.

772

A. Czopek et al.

(7)

Figure 4Macrophages clear endothelin-1 through endothelin-B receptor-mediated endocytosis. Endothelin-1 concentration in cell culture super-natant from bone marrow-derived M/ (A) over 24 h after incubation with a starting endothel1 concentration of 10 pg/mL, (B) at 24 h following in-cubation with endothelin-1 (10–104pg/mL), (C) after incubation with an endothelin-A (BQ123) or endothelin-B (BQ788) antagonist or dynasore, an inhibitor of dynamin-dependent endocytosis receptor antagonism, and (D) following bone marrow-derived M/ endothelin-B receptor knockdown with siRNA (efficiency of knockdown left panel). Kruskal–Wallis tests were used with Dunn’s test for multiple planned comparisons. Adjusted P-val-ues are shown. For (D) left panel an unpaired t-test was used; the two-tailed P-value is shown.

Continued

773

(8)

Figure 4 (E) Vascular smooth muscle cells do not remove endothelin-1 from their media in vitro. Data from individual experiments are shown and were compared by Kruskal–Wallis; with Dunn’s test for multiple planned comparisons as shown. (F) Wild type (white circles) and LysMETB-/-(black

circles) bone marrow-derived M/ were exposed to endothelin-1 10 pg/mL in vitro in the presence or absence of BQ788, an endothelin-B receptor antagonist. Endothelin-1 was measured in the supernatant at 24 h. One-way analysis of variance (P = 0.0003) was used and adjusted P-values for mul-tiple comparisons are shown. (G) Uptake of fluorescent endothelin-1 by wild type or LysMETB-/-bone marrow-derived M/ in the presence or

ab-sence of BQ788. One-way analysis of variance (P < 0.0001) was used and adjusted P-values for planned comparisons, as shown. (H) Rapid uptake of endothelin-1 by bone marrow-derived M/.

774

A. Czopek et al.

(9)

Figure 5 Macrophages modify vascular contraction and oxidative stress in response to endothelin-1. (A) Contraction of mesenteric artery seg-ments to increasing (endothelin-1) (Control, black line, n = 10) and following pre-incubation with varying number of M/ (n = 6 per group). (B) Vascular responses to 107M/ (red line, n = 6), M/þ BQ123 (dotted red line, n = 4), M/ from LysMETB-/-mice (blue line, n = 4), and M/þ BQ788

(dotted blue line, n = 6). Data (mean ± standard deviation) were compared by two-way analysis of variance, with main effects of endothelin-1, pre-treatment and the interaction (all P < 0.0001). Adjusted P-values for planned comparisons are shown and only the comparisons where P < 0.05 at 107_{endothelin-1 are shown. (C) The effects of M/ on oxidative stress. Mean ± standard deviation were compared by one-way analysis of variance}

(P = 0.0002), using Sidak correction for multiple comparisons, with adjusted P-values are shown.

775

(10)

..

.

was approximately two-fold greater in M/ deficient mice compared with control groups. Notably, M/ depletion did not affect the acute pressor response to ANG II (Figure6D).

To specifically explore the role of the M/ ETBreceptor in the

pressor response to ET-1, we administered i.v. ET-1 to LysMETB

-/-and control mice. Baseline MAP did not differ between the two groups (Figure6E). Similar to the response seen following systemic M/ depletion, LysMETB-/-mice demonstrated an exaggerated pressor

response to ET-1 (Figure6F). At a dose of ET-1 0.1 nmol/kg the max-imal change in MAP was approximately two-fold that seen in control animals.

Macrophage depletion augments the

chronic hypertensive response to

endothelin-1 and adoptive transfer of

wild type monocytes prevents this

A 3-week infusion of ET-1 led to sustained increases in systolic and diastolic BP in CD11b-DTR mice (Figure7A and B). Both systolic and diastolic BP increased by approximately 10–12 mmHg above base-line. DT was used to deplete M/ over a period of 7–10 days resulting in gradual increases in both systolic and diastolic BP (Figure7C and D). As the effects of DT weaned, and M/ repopulated, BP gradually returned to pre-depletion levels. The maximal increase in systolic BP was approximately 20 mmHg and for diastolic BP this was approxi-mately 15 mmHg. In the negative control arm, CD11b-DTR mice were injected with phosphate buffered saline, and here, BP remained stable throughout the experiment.

In keeping with an important role for circulating monocytes in these effects, adoptive transfer of wild type monocytes prevented the rise in BP seen with DT (Figure7E and F). As might be expected, M/ depletion was associated with a rise in circulating ET-1 with a fall to pre-depletion levels with M/ repopulation (Figure7G).

Macrophage depletion augments the

chronic hypertensive response to a high

salt diet

Next, we explored the role of M/ in a second model of hypertension that associated with a high salt diet. High salt led to rises in both sys-tolic and diassys-tolic BP of approximately 10 mmHg above baseline (see

Supplementary material online,Figure S13A). M/ depletion led to fur-ther rises in both and, as previously seen, these effects diminished with M/ repopulation (see Supplementary material online,Figure S13Band C).

Myeloid ET

B

receptor deficiency

augments the chronic hypertensive

response to endothelin-1 and

angiotensin II

In line with an important role for the myeloid ETBreceptor in

pro-tecting from the deleterious effects of ET-1, ET-1 administration into LysMETB-/-mice led to a two- to three-fold exaggerated hypertensive

response to ET-1 compared with littermate control mice (Figure8A). To confirm our findings, we exposed LysMETB-/-and littermate

con-trol mice to a 2-week infusion of ANG II, a model of hypertension that is ET-1 dependent.20,21Baseline systolic and diastolic BP were

similar between the two groups of animals during both the active (night) and inactive (day) phases. LysMETB

-/-mice had a significantly exaggerated hypertensive response to ANG II compared with their controls. For example, at night, systolic BP rose on average by ap-proximately 25 mmHg in control mice but by apap-proximately 40 mmHg in knockouts. For diastolic BP, these corresponding figures were approximately 25 mmHg and approximately 35 mmHg (Figure8B).

In patients with anti-neutrophil

cytoplasmic antibody vasculitis different

immunotherapies variably affect blood

pressure and the endothelin system

Cyclophosphamide (CYC) and mycophenolate mofetil (MMF) are standard therapies for patients with small vessel vasculitis associated with autoantibodies to neutrophil cytoplasmic antigens (ANCA). Cyclophosphamide not only depletes B and T cells but also effectively depletes circulating and tissue M/28; MMF suppresses T- and B-cell function but does not deplete M/. Thus, we investigated changes in BP and the ET system following treatment in 20 patients with ANCA vasculitis: 10 received CYC as their immunosuppressive therapy whereas the other 10 received MMF. Demographics and other treat-ments, including corticosteroid dose, antihypertensive treatment, and the use of plasmapharesis, were similar between groups (see

Supplementary material online,Table S2).

Baseline systolic and diastolic BP, plasma and urine ET-1 did not dif-fer between the two groups (see Supplementary material online,

Table S2). Interestingly, plasma ET-1 was higher in patients with ANCA vasculitis than in healthy volunteers (3.74 ± 0.38 pg/mL vs. 1.21 ± 1.2 pg/mL, P = 0.02). Whereas CYC reduced mean circulating monocyte count MMF did not (baseline vs. week 6: CYC: 0.87 ± 0.15 vs. 0.26 ± 0.17 109

, P < 0.05; MMF: 0.77 ± 0.25 vs. 0.69 ± 0.29 109

, P = 0.756). Cyclophosphamide treatment was associated with a greater increase in BP compared with treatment with MMF (Figure9A and B, P < 0.01 for CYC vs. MMF). There was a positive correlation between the change in peripheral monocyte count and the extent to which BP rose in those patients receiving CYC (Figure9C and D). Plasma ET-1 fell following treatment with MMF (Figure9E, P < 0.001 for both weeks 0 vs. 6 and 6 vs. 12), whereas CYC treatment only led to an initial fall in plasma 1 (P < 0.05 for weeks 0 vs. 6). Plasma ET-1 was lower at week ET-12 in those treated with MMF (Figure9E). Urine ET-1 fell with both CYC and MMF (Figure9F, P < 0.01 for CYC and P < 0.0001 for MMF for weeks 0 vs. 12) but to a greater degree with MMF (P < 0.01 for MMF vs. CYC). Flow-mediated dilation (FMD) of the brachial artery was similar at baseline between the two groups (6.7 ± 0.2 for MMF vs. 6.6 ± 0.1% for CYC, P = 0.589). At 12 weeks, FMD had not changed in those patients receiving MMF treatment) whereas those patients receiving CYC had lower FMD in keeping with worse endothelial function (-1.2%, P < 0.05 vs. baseline).

In these same patients, we characterized M/ expression of ETA

and ETBreceptors and compared this to health (seeSupplementary

material online,Table S2andFigure S9Gand H). There were no differ-ences in expression of the ETAreceptor between the groups and in

keeping with our previous data its expression was lower than that of the ETBreceptor. Interestingly, patients presenting with ANCA

vas-culitis demonstrated reduced expression of the ETBreceptor on

776

A. Czopek et al.

(11)

Figure 6 Role of M/ in the pressor response to endothelin-1. Acute blood pressure response to incremental doses of endothelin-1 following acute depletion of circulating monocytes and resident M/ (DTRþDTþ), and in controls [those with the diphtheria toxin receptor (DTR) construct but given saline, DTRþDT-, and mice given diphtheria toxin (DT), DTR-DTþ; n = 10 mice/group]. (A) Baseline blood pressure. (B) Example of the differences seen among the three groups in the acute pressor response to endothelin-1. Intravenous endothelin-1 administration is defined by the black arrow. The dotted and dashed lines represent baseline and maximal mean arterial pressure, respectively. (C) Maximal change in mean arterial pressure, compared by two-way analysis of variance (main effect of genotype, of endothelin-1 dose and the interaction all P = 0.0001); Holm–Sidak planned comparisons were made and adjusted P-values are shown. (D) Acute blood pressure response to ANG II (1 nmol/kg) following depletion of circulating monocytes and resident M/. (E) Baseline mean arterial pressure and (F) maximal change in mean arterial pressure in response to endothelin-1 in LysMETB-/-and littermate control mice (n = 6 mice per group), compared by two-way analysis of variance

[main effect of genotype P = 0.0128, of endothelin-1 dose (P = 0.0001) and the interaction P = 0.0004]; Holm–Sidak planned comparisons were made and adjusted P-values are shown.

777

(12)

Figure 7Effects of chronic M/ depletion on endothelin-1-mediated hypertension. Effects on systolic and diastolic blood pressure of endothelin-1 infusion (10 pmol/kg/min administered via minipump) for 3 weeks (mean ± standard deviation, n = 12 mice, A and B). From Day 21, half the mice (n = 6) underwent M/ depletion over the period shown in grey. Groups were compared by two-away analysis of variance, assessing the main effect of M/ depletion, duration in days and the interaction. The effect of M/ depletion was P < 0.0001 for (C) systolic blood pressure and (D) diastolic blood pressure. Effects of diphtheria toxin alone or diphtheria toxin with adoptive transfer of CD11bþGr1þmonocytes at Day 21 and Day 24 on sys-tolic and diassys-tolic blood pressure are shown in (E) and (F). Two-way analysis of variance reported a significant (P = 0.0003) effect of group. (G) The effects of M/ depletion and repopulation on plasma endothelin-1 (n = 5 mice per group), assessed by one-way analysis of variance (P < 0.0001), with the adjusted P-values for planned comparisons are shown.

778

A. Czopek et al.

(13)

Figure 8 Effects of myeloid endothelin-B receptor deficiency on endothelin-1 and angiotensin II-mediated hypertension. Night-time and daytime telemetry systolic and diastolic blood pressure in LysMETB-/-and wild type controls receiving 2 weeks of endothelin-1 (5 pmol/kg/min; n = 8 mice per

group) (A) or angiotensin II (1 mg/kg/min; n = 12 mice/group) (B). Data are mean ± standard deviation and two-way analysis of variance compared the main effects of genotype, treatment (endothelin-1 or angiotensin II) and the interaction. In all cases, there was a significant (P<0.0001) effect of genotype.

779

(14)

Figure 9Effects of treatment with cyclophosphamide and mycophenolate mofetil in patients with autoantibodies to neutrophil cytoplasmic anti-gens vasculitis. (A and B) Systolic and diastolic blood pressure, with two-way analysis of variance showing a significant effect of treatment (P = 0.001). (C and D) The relationship between change in circulating monocyte count and changes in systolic and diastolic blood pressure is shown. (E) Plasma endothelin-1 (effect of treatment P = 0.0225) and (F) urine endothelin-1 (effect of treatment P = 0.2053). (G and H) Monocyte expression of endothe-lin-A (P = 0.1310) and endothelin-B receptors (P = 0.0001) in patients with autoantibodies to neutrophil cytoplasmic antigens vasculitis, compared by one-way analysis of variance with adjusted P-values are shown.

780

A. Czopek et al.

(15)

..

.

their M/ compared with levels seen in health. This was normalized by treatment with MMF but further reduced following treatment with CYC.

Discussion

For the first time, we have demonstrated that the M/ ETBreceptor

in both mouse and humans provides a novel clearance mechanism for ET-1. Its functional importance in vivo is demonstrated by the exaggerated pro-hypertensive effect of ET-1 and ANG II in mice with a deletion of the M/ ETBreceptor or following systemic M/

deple-tion. Interestingly, and unexpectedly, we found no evidence that ET-1 was able to polarize mouse or human M/ towards a classical pro-inflammatory or alternative anti-pro-inflammatory phenotype but both displayed chemokinesis towards ET-1. Overall, these data provide us with new knowledge, and a clarification of mechanisms underlying the pathological basis of hypertension in relation to the immune and ET systems (Figure10).

To date, many of the studies investigating the role of the innate im-mune system in the pathogenesis of, and response to, hypertension have focused on the role of T cells in relation to ANG II-mediated hypertension.29–31Few have examined the role of M/ and only one study relates to ET-1-mediated vascular injury.32Recent studies have explored the effects of altering the phenotype of bone marrow-derived cells9or M/10on hypertension and its complications, where-as others have elegantly depleted neutrophils and M/8to this end.

The results of these studies are often contradictory but, nevertheless, suggest that M/ may contribute to, and protect from, hypertension. The study by Machnik et al.7showed that salt loading increases M/ accumulation in the subcutaneous space. These M/ are stimulated by the hypertonic environment to produce vascular endothelial growth factor C. This leads to proliferation of lymphatics. This is pro-tective, because clodronate-mediated M/ depletion prevents the lymphatic proliferation and leads to hypertension in response to salt loading. These landmark findings support a role for M/ acting as a buffering mechanism, protecting against the development of hyper-tension. Our study extends beyond these, providing a mechanistic understanding of the interaction between M/ and local vascular con-tractility and the impact on BP.

The M/ system comprises a spectrum of cell types, and depletion strategies vary in their specificity and efficacy.33Hence, we used two depletion models here, the CD11b-DTR (reduction in M/ number) and lysozyme M (LysM; alternation in M/ phenotype)-Cre systems. DT administration on CD11b-DTR mice achieved 90% ablation of circulating monocytes. In terms of resident cells, there was a significant reduction in renal M/ but less of an effect in the liver and spleen. Analysis of the main monocyte subsets (Gr11 CCR21 CX3CR12 and Gr12 CCR22 CX3CR11) demonstrated that both are equally depleted over the time course of our studies. DT administration did not deplete neutrophils. These findings are in keeping with earlier studies.34,35Using the LysM-Cre system, there is functional depletion (80–100%) of mature M/ but also neutrophils. Figure 10 Macrophage-endothelin system interplay. Endothelin-1 is produced predominantly by vascular endothelial cells. EDN1 gene transcrip-tion produces pre-pro endothelin-1 which is cleaved to big endothelin-1 and then endothelin-1. Endothelin-1 is largely secreted abluminally where binding to endothelin-A and endothelin-B receptors on vascular smooth muscle cells causes vasoconstriction. Endothelin-B receptor activation on endothelial cells results in the release of prostacyclin and NO and consequently vasodilatation. Macrophages express both endothelin-A and endo-thelin-B receptors and display chemokinesis towards endothelin-1. However, endothelin-1 does not polarize M/ to a pro-inflammatory or anti-in-flammatory phenotype. Our data suggest that M/ clear endothelin-1 through endothelin-B receptor-mediated uptake. Binding of endothelin-1 to endothelin-B receptor on M/ results in dynamin-dependent endocytosis. This endothelin-B receptor bound endothelin-1 is then transported to the lysosomes for degradation.

781

(16)

Take home ﬁgureA novel role for myeloid endothelin-B receptors in hypertension.

782

A. Czopek et al.

(17)

..

.

Additionally, this system may partially (16–20%) deplete dendritic cells (DCs),36an effect that is more pronounced in the CD11b-DTR mice.35

Both CD11b-DTR mice administered DT and LysMETB-/- mice

were more sensitive to the pressor effects of ET-1. Taken together it is likely these effects were due to an absence of circulating mono-cytes and/or vascular M/. Given the acute nature of the response, we hypothesize that in control mice circulating monocytes remove the intravenously administered ET-1 to some extent thus attenuating the amount reaching the target VSMC. In contrast, the absence of this ET-1 clearance in M/-deplete mice allows more to be available to act on these cells promoting an exaggerated vasoconstrictor and so hypertensive response. There may be a contribution from DCs and neutrophils to these effects. Although there is currently no evi-dence that murine or human DCs are able to regulate ET-1, by its clearance or degradation, one study has shown that DCs are able to synthesize ET-1 in response to inflammatory stimuli37although an-other has suggested the opposite.38Interestingly, the few data on neutrophils and ET-1 suggest that these cells can both produce and degrade ET-1.39

Our data also demonstrate that M/ are important in hypertension. We used three models of hypertension—chronic ET-1 and ANG II infusion, which are both dependent on the ET system,20,21,40,41_{and a}

high salt diet. M/ depletion with repeated doses of DT resulted in gradual increases in both systolic and diastolic BP in chronic ET-1 and salt-dependent hypertension. Interestingly, once the DT administra-tion was stopped (and M/ were allowed to repopulate) as well as monocyte adoptive transfer resulted in both systolic and diastolic BP returning to pre-depletion levels. This suggests that the importance of both circulating and/or organ-based M/ and future work should focus on discriminating which of these is more important here. The importance of the ETBreceptor in chronic hypertension is provided

by data from our mice genetically deficient for ETBon M/ alone.

Here, two separate models of hypertension, chronic ET-1 and chron-ic ANG II infusion, elchron-icited exaggerated rises in both systolchron-ic and dia-stolic BP in knockout animals compared with littermate controls.

ET-1 is considered to be pro-inflammatory, so it was surprising that it was unable to polarize M/ phenotype, at least in vitro. This was true at a range of ET-1 concentrations (10–10 000 pg/mL; mean plasma ET-1 in mice 2–3 pg/mL42), whether the ET-1 was adminis-tered prior to, following or concomitantly with classical (LPS/INFc) or alternative (IL-4/IL-13) stimulation. The few data supporting a pro-inflammatory effect of ET-1 on M/ used immortalized M/ tumour cell lines and lacked robust methodology.43,44 Interestingly, and in keeping with earlier studies,37,45LPS/INFc stimulation of mouse M/ did increase ET-1 concentrations in the supernatant at 24 h and this was completely blocked by an inhibitor of ECE suggesting that this increase in immunoreactive ET-1 is a result of de novo production by M/.

Mouse BMDM demonstrated chemokinesis towards ET-1 and this was reduced by selective ETAantagonism and completely abrogated

by selective ETBblockade. Two recent studies support our

find-ings46,47but both found that the ability of M/ to move towards ET-1 was more dependent on the ETAreceptor than the ETB. Of note,

both studies investigated that the role of M/ and the ET system in the setting of cancer (bladder and breast) where there may well be several different M/ phenotypes with a different balance of ETA:ETB

receptors. In support of ETB-mediated chemokinesis, BMDM from

LysMETB-/-displayed no migration towards ET-1. This was not due to

an inability to move as they retained their chemokinetic response to MCP-1. Our data showing that M/ produce ET-1 in response to an inflammatory stimulus allows us to postulate that this may in turn lead to recruitment of further M/ to the area of inflammation as a mechanism to propagate or regulate the response of the innate im-mune system.

Thus, although M/ are not activated by ET-1 they migrate towards it and clear the peptide through ETBreceptor-mediated uptake

pro-viding a novel clearance mechanism for the peptide. In EC, the ETB

receptor resides within caveolae. Binding of ET-1 to endothelial ETB

stimulates rapid budding and internalization of the caveolae contain-ing the ETBreceptor bound ET-1.

48

This mechanism is dynamin-dependent. In our in vitro studies, both mouse and human M/ removed ET-1 from their surrounding media, an effect that was sig-nificantly reduced by selective antagonism (or knockdown) of the ETBreceptor but unaffected by ETAblockade. In keeping with ET-1

clearance by caveolar ETBreceptors, inhibiting dynamin GTPase

ac-tivity with dynasore completely prevented ET-1 removal by M/. M/ are multi-functional cells and are able to degrade peptides through the secretion of proteases as well as through the activity of the cell surface metalloprotease, NEP.49_{However, broad protease and NEP}

inhibition did not affect M/ ET-1 uptake.

To demonstrate the clinical relevance of our findings, we studied patients with ANCA-associated vasculitis, a potentially life-threatening autoimmune condition. Circulating ET-1 was higher in those with vasculitis than in health, probably contributed to by sys-temic inflammation and endothelial dysfunction. Both MMF and CYC are standard therapies for this condition but they differ in their mech-anisms of action. Whereas, MMF inhibits T- and B-cell proliferation and function,50CYC is directly cytotoxic and depletes not only these cells but also circulating and tissue M/.28In keeping with this action, we demonstrate that CYC has a tendency to reduce the circulating monocyte count by approximately 50% whereas MMF does not. Although this is an accepted measure of circulating monocytes51it does not account for tissue-based M/. Nevertheless, we show here that those patients receiving CYC have a greater increase in BP and deterioration in endothelial function than those receiving MMF. In part, this may be due to loss of M/-ET regulation. This is supported by the greater fall in plasma ET-1 in the MMF, but not CYC group, and the down-regulation of the M/ ETBreceptor with CYC but not

MMF. Thus, hypertension in CYC-treated patients may respond well to ET antagonists. This hypothesis should be explored in future clinic-al studies as there are few data that relate to the impact of vasculitis and its treatment on BP or vascular function—which is important be-cause ET antagonism may have broader cardiovascular benefits.52–55

In summary, our study has identified a new interaction between M/ and the endothelin system, whereby M/ are drawn to ET-1, without any evident effect on polarization of phenotype, and clear ET-1 from the surrounding milieu. In vivo, this cellular action has a sig-nificant impact on acute vascular function and BP; importantly this pathway exerts a restraining effect on BP in chronic hypertension. Our final study strongly suggests that this system is operational in humans and therefore represents an intriguing opportunity to modu-late BP and reduce cardiovascular risk in multi-system inflammatory conditions. In future, studies such as ours might lead to newer and

783

(18)

..

.

currently available BP lowering treatments being used in a more ra-tional way for specific groups of patients, such as those with cancer, as outlined in recent guidelines.56

Supplementary material

Supplementary materialis available at European Heart Journal online.

Acknowledgements

The authors would like to thank Anthony Davenport for the antisera against the endothelin-A and endothelin-B receptors.

Funding

This work was supported by the British Heart Foundation [FS/11/78/ 29328; FS/13/30/29994; FS/16/54/32730], Kidney Research UK [IN10/ 2010], and the Institut National de Sante´ et de la Recherche Me´dicale (INSERM) and research grant from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ ERC grant agreement no 107037 (to Dr. Tharaux).

Conflict of interest: none declared.

References

1. Lewington S, Clarke R, Qizilbash N, Peto R, Collins R. Age-specific relevance of usual blood pressure to vascular mortality: a meta-analysis of individual data for one million adults in 61 prospective studies. Lancet 2002;360:1903–1913. 2. Kearney PM, Whelton M, Reynolds K, Muntner P, Whelton PK, He J. Global

bur-den of hypertension: analysis of worldwide data. Lancet 2005;365:217–223. 3. Verlohren S, Muller DN, Luft FC, Dechend R. Immunology in hypertension,

pre-eclampsia, and target-organ damage. Hypertension 2009;54:439–443.

4. Clozel M, Kuhn H, Hefti F, Baumgartner HR. Endothelial dysfunction and suben-dothelial monocyte macrophages in hypertension. Effect of angiotensin convert-ing enzyme inhibition. Hypertension 1991;18:132–141.

5. Franco M, Martinez F, Quiroz Y, Galicia O, Bautista R, Johnson RJ, Rodriguez-Iturbe B. Renal angiotensin II concentration and interstitial infiltration of immune cells are correlated with blood pressure levels in salt-sensitive hypertension. Am J Physiol 2007;293:R251–R256.

6. Rodriguez-Iturbe B, Vaziri ND, Johnson RJ. Inflammation, angiotensin II, and hypertension. Hypertension 2008;52:e135; author reply e136.

7. Machnik A, Neuhofer W, Jantsch J, Dahlmann A, Tammela T, Machura K, Park JK, Beck FX, Muller DN, Derer W, Goss J, Ziomber A, Dietsch P, Wagner H, van Rooijen N, Kurtz A, Hilgers KF, Alitalo K, Eckardt KU, Luft FC, Kerjaschki D, Titze J. Macrophages regulate salt-dependent volume and blood pressure by a vascular endothelial growth factor-C-dependent buffering mechanism. Nat Med 2009;15:545–552.

8. Wenzel P, Knorr M, Kossmann S, Stratmann J, Hausding M, Schuhmacher S, Karbach SH, Schwenk M, Yogev N, Schulz E, Oelze M, Grabbe S, Jonuleit H, Becker C, Daiber A, Waisman A, Munzel T. Lysozyme M-positive monocytes mediate angiotensin II-induced arterial hypertension and vascular dysfunction. Circulation 2011;124:1370–1381.

9. Crowley SD, Song YS, Sprung G, Griffiths R, Sparks M, Yan M, Burchette JL, Howell DN, Lin EE, Okeiyi B, Stegbauer J, Yang Y, Tharaux PL, Ruiz P. A role for angiotensin II type 1 receptors on bone marrow-derived cells in the pathogenesis of angiotensin II-dependent hypertension. Hypertension 2010;55:99–108. 10. Rickard AJ, Morgan J, Tesch G, Funder JW, Fuller PJ, Young MJ. Deletion of

mineralocorticoid receptors from macrophages protects against

deoxycorticosterone/salt-induced cardiac fibrosis and increased blood pressure. Hypertension 2009;54:537–543.

11. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T. A novel potent vasoconstrictor peptide produced by vascu-lar endothelial cells. Nature 1988;332:411–415.

12. Boesen EI, Sasser JM, Saleh MA, Potter WA, Woods M, Warner TD, Pollock JS, Pollock DM. Interleukin-1beta, but not interleukin-6, enhances renal and system-ic endothelin production in vivo. Am J Physiol 2008;295:F446–F453.

13. Sprague AH, Khalil RA. Inflammatory cytokines in vascular dysfunction and vascu-lar disease. Biochem Pharmacol 2009;78:539–552.

14. Arai H, Hori S, Aramori I, Ohkubo H, Nakanishi S. Cloning and expression of a cDNA encoding an endothelin receptor. Nature 1990;348:730–732.

15. Sakurai T, Yanagisawa M, Takuwa Y, Miyazaki H, Kimura S, Goto K, Masaki T. Cloning of a cDNA encoding a non-isopeptide selective subtype of the endothe-lin receptor. Nature 1990;348:732–735.

16. Davenport AP, O’Reilly G, Molenaar P, Maguire JJ, Kuc RE, Sharkey A, Bacon CR, Ferro A. Human endothelin receptors characterized using reverse transcriptase-polymerase chain reaction, in situ hybridization, and subtype-selective ligands BQ123 and BQ3020: evidence for expression of ETB receptors in human vascu-lar smooth muscle. J Cardiovasc Pharmacol 1993;22:S22–S25.

17. Verhaar MC, Strachan FE, Newby DE, Cruden NL, Koomans HA, Rabelink TJ, Webb DJ. Endothelin-A receptor antagonist-mediated vasodilatation is attenu-ated by inhibition of nitric oxide synthesis and by endothelin-B receptor block-ade. Circulation 1998;97:752–756.

18. Newby DE, Strachan FE, Webb DJ. Abnormal endothelin B receptor vasomotor responses in patients with Hirschprung’s disease. QJM 2002;95:159–163. 19. Schiffrin EL. State-of-the-Art lecture. Role of endothelin-1 in hypertension.

Hypertension 1999;34:876–881.

20. Rajagopalan S, Laursen JB, Borthayre A, Kurz S, Keiser J, Haleen S, Giaid A, Harrison DG. Role for endothelin-1 in angiotensin II-mediated hypertension. Hypertension 1997;30:29–34.

21. Moreau P, d’Uscio LV, Shaw S, Takase H, Barton M, Lu¨Scher TF. Angiotensin II

increases tissue endothelin and induces vascular hypertrophy. Reversal by ETA

-receptor antagonist. Circulation 1997;96:1593–1597.

22. Krum H, Viskoper RJ, Lacourciere Y, Budde M, Charlon V. The effect of an endothelin-receptor antagonist, bosentan, on blood pressure in patients with es-sential hypertension. N Engl J Med 1998;338:784–790.

23. Nakov R, Pfarr E, Eberle S. Darusentan: an effective endothelin A receptor antag-onist for treatment of hypertension. Am J Hypertens 2002;15:583–589. 24. Weber MA, Black H, Bakris G, Krum H, Linas S, Weiss R, Linseman JV, Wiens

BL, Warren MS, Lindholm LH. A selective endothelin-receptor antagonist to re-duce blood pressure in patients with treatment-resistant hypertension: a rando-mised, double-blind, placebo-controlled trial. Lancet 2009;374:1423–1431. 25. Boesen EI, Krishnan KR, Pollock JS, Pollock DM. ETA activation mediates

angio-tensin II-induced infiltration of renal cortical T cells. J Am Soc Nephrol 2011;22: 2187–2192.

26. Tostes RC, Touyz RM, He G, Ammarguellat F, Schiffrin EL. Endothelin A recep-tor blockade decreases expression of growth facrecep-tors and collagen and improves matrix metalloproteinase-2 activity in kidneys from stroke-prone spontaneously hypertensive rats. J Cardiovasc Pharmacol 2002;39:892–900.

27. Sasser JM, Sullivan JC, Hobbs JL, Yamamoto T, Pollock DM, Carmines PK, Pollock JS. Endothelin A receptor blockade reduces diabetic renal injury via an anti-inflammatory mechanism. J Am Soc Nephrol 2007;18:143–154.

28. Santosuosso M, Divangahi M, Zganiacz A, Xing Z. Reduced tissue macrophage population in the lung by anticancer agent cyclophosphamide: restoration by local granulocyte macrophage-colony-stimulating factor gene transfer. Blood 2002;99:1246–1252.

29. Barhoumi T, Kasal DA, Li MW, Shbat L, Laurant P, Neves MF, Paradis P, Schiffrin EL. T regulatory lymphocytes prevent angiotensin II-induced hypertension and vascular injury. Hypertension 2011;57:469–476.

30. Zhang JD, Patel MB, Song YS, Griffiths R, Burchette J, Ruiz P, Sparks MA, Yan M, Howell DN, Gomez JA, Spurney RF, Coffman TM, Crowley SD. A novel role for type 1 angiotensin receptors on T lymphocytes to limit target organ damage in hypertension. Circ Res 2012;110:1604–1617.

31. Guzik TJ, Hoch NE, Brown KA, McCann LA, Rahman A, Dikalov S, Goronzy J, Weyand C, Harrison DG. Role of the T cell in the genesis of angiotensin II induced hypertension and vascular dysfunction. J Exp Med 2007;204:2449–2460. 32. Javeshghani D, Barhoumi T, Idris-Khodja N, Paradis P, Schiffrin EL. Reduced

macrophage-dependent inflammation improves endothel1-induced vascular in-jury. Hypertension 2013;62:112–117.

33. Chow A, Brown BD, Merad M. Studying the mononuclear phagocyte system in the molecular age. Nat Rev Immunol 2011;11:788–798.

34. Duffield JS, Forbes SJ, Constandinou CM, Clay S, Partolina M, Vuthoori S, Wu S, Lang R, Iredale JP. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. J Clin Invest 2005;115:56–65.

35. Ferenbach DA, Sheldrake TA, Dhaliwal K, Kipari TM, Marson LP, Kluth DC, Hughes J. Macrophage/monocyte depletion by clodronate, but not diphtheria toxin, improves renal ischemia/reperfusion injury in mice. Kidney Int 2012;82:928–933. 36. Clausen BE, Burkhardt C, Reith W, Renkawitz R, Forster I. Conditional gene

tar-geting in macrophages and granulocytes using LysMcre mice. Transgen Res 1999; 8:265–277.

37. Spirig R, Potapova I, Shaw-Boden J, Tsui J, Rieben R, Shaw SG. TLR2 and TLR4 agonists induce production of the vasoactive peptide endothelin-1 by human dendritic cells. Mol Immunol 2009;46:3178–3182.

38. Slobodin G, Pavlotzky E, Panov J, Rosner I, Kessel A, Toubi E. Endothelin-1 does not change the function of monocyte-derived dendritic cells grown from patients with systemic sclerosis. Immunol Invest 2008;37:841–848.

784

A. Czopek et al.

(19)

..

39. Sessa WC, Kaw S, Zembowicz A, Anggard E, Hecker M, Vane JR. Human poly-morphonuclear leukocytes generate and degrade endothelin-1 by two distinct neutral proteases. J Cardiovasc Pharmacol 1991;17:S34–S38.

40. Elmarakby AA, Loomis ED, Pollock JS, Pollock DM. NADPH oxidase inhibition attenuates oxidative stress but not hypertension produced by chronic ET-1. Hypertension 2005;45:283–287.

41. Saleh MA, Boesen EI, Pollock JS, Savin VJ, Pollock DM. Endothelin-1 increases glomerular permeability and inflammation independent of blood pressure in the rat. Hypertension 2010;56:942–949.

42. Bagnall AJ, Kelland NF, Gulliver-Sloan F, Davenport AP, Gray GA, Yanagisawa M, Webb DJ, Kotelevtsev YV. Deletion of endothelial cell endothelin B receptors does not affect blood pressure or sensitivity to salt. Hypertension 2006;48:286–293. 43. Ruetten H, Thiemermann C. Endothelin-1 stimulates the biosynthesis of tumour

necrosis factor in macrophages: ET-receptors, signal transduction and inhibition by dexamethasone. J Physiol Pharmacol 1997;48:675–688.

44. Wilson SH, Simari RD, Lerman A. The effect of endothelin-1 on nuclear factor kappa B in macrophages. Biochem Biophys Res Commun 2001;286:968–972. 45. Ehrenreich H, Anderson RW, Fox CH, Rieckmann P, Hoffman GS, Travis WD,

Coligan JE, Kehrl JH, Fauci AS. Endothelins, peptides with potent vasoactive proper-ties, are produced by human macrophages. J Exp Med 1990;172:1741–1748. 46. Said N, Smith S, Sanchez-Carbayo M, Theodorescu D. Tumor endothelin-1

enhances metastatic colonization of the lung in mouse xenograft models of blad-der cancer. J Clin Invest 2011;121:132–147.

47. Chen CC, Chen LL, Hsu YT, Liu KJ, Fan CS, Huang TS. The endothelin-integrin axis is involved in macrophage-induced breast cancer cell chemotactic interac-tions with endothelial cells. J Biol Chem 2014;289:10029–10044.

48. Oh P, Horner T, Witkiewicz H, Schnitzer JE. Endothelin induces rapid, dynamin-mediated budding of endothelial caveolae rich in ET-B. J Biol Chem 2012;287: 17353–17362.

49. Schreiter A, Gore C, Labuz D, Fournie-Zaluski MC, Roques BP, Stein C, Machelska H. Pain inhibition by blocking leukocytic and neuronal opioid pepti-dases in peripheral inflamed tissue. FASEB J 2012;26:5161–5171.

50. Ransom JT. Mechanism of action of mycophenolate mofetil. Ther Drug Monitor 1995;17:681–684.

51. Barr LC, Brittan M, Morris AC, McAuley DF, McCormack C, Fletcher AM, Richardson H, Connell M, Patel D, Wallace WA, Rossi AG, Davidson DJ, Manson L, Turner M, Hirani N, Walsh TS, Anderson NH, Dhaliwal K, Simpson AJ. A randomized controlled trial of peripheral blood mononuclear cell depletion in experimental human lung inflammation. Am J Resp Crit Care Med 2013;188: 449–455.

52. Dhaun N, Goddard J, Kohan DE, Pollock DM, Schiffrin EL, Webb DJ. Role of endothelin-1 in clinical hypertension: 20 years on. Hypertension 2008;52:452–459. 53. Dhaun N, Goddard J, Webb DJ. The endothelin system and its antagonism in

chronic kidney disease. J Am Soc Nephrol 2006;17:943–955.

54. Dhaun N, Johnston NR, Goddard J, Webb DJ. Chronic selective endothelin a re-ceptor antagonism reduces serum uric Acid in hypertensive chronic kidney dis-ease. Hypertension 2011;58:e11–e12.

55. Dhaun N, MacIntyre IM, Kerr D, Melville V, Johnston NR, Haughie S, Goddard J, Webb DJ. Selective endothelin-A receptor antagonism reduces proteinuria, blood pressure, and arterial stiffness in chronic proteinuric kidney disease. Hypertension 2011;57:772–779.

56. Williams B, Mancia G, Spiering W, Agabiti Rosei E, Azizi M, Burnier M, Clement DL, Coca A, de Simone G, Dominiczak A, Kahan T, Mahfoud F, Redon J, Ruilope L, Zanchetti A, Kerins M, Kjeldsen SE, Kreutz R, Laurent S, Lip GYH, McManus R, Narkiewicz K, Ruschitzka F, Schmieder RE, Shlyakhto E, Tsioufis C, Aboyans V, Desormais I; ESC Scientific Document Group. 2018 ESC/ESH Guidelines for the management of arterial hypertension. Eur Heart J 2018;39:3021–3104.

784a

A. Czopek et al.